Project Summary/Abstract The mitochondrial respiratory chain (RC) is the central apparatus enabling mitochondria to undergo oxidative phosphorylation (OXPHOS) and supply cellular energy. Defective RCs result in a variety of potentially devastating inborn errors of metabolism with no proven effective treatment. Additionally, aging and many common age-related diseases including cancer, type 2 diabetes, Alzheimer?s and Parkinson?s diseases also exhibit notable decreases in RC activity. Recent findings from our laboratory show that cells have an innate capability for coping with defective RCs that is only accessible upon activation of the hypoxia response, a genetic program evolved to adapt cells to limited oxygen availability. Growth defects in cultured cells treated with RC inhibitors are rescued with activation of the hypoxia response. Strikingly, this rescue is recapitulated in a genetic mouse model of pediatric mitochondrial disease featuring a dysfunctional RC, attesting to the generalizability of this effect. Mice lacking a key complex I subunit within the RC develop a fatally progressive encephalopathy and die after ~60 days exposure to normal oxygen levels, but neurodegeneration is prevented and even reversed after chronic exposure to hypoxia, with longevity increased to ~ 270 days. As the hypoxia response upregulates hundreds of individual genes, mechanistically it remains unclear which genes and what processes defend a cell against RC dysfunction. This proposal aims to identify the molecular players within the hypoxia response that enable cells to cope with a defective RC. The first specific aim will explore the genetic effectors of the hypoxia rescue of RC dysfunction by both targeted and non-targeted approaches, examining the rescue effect in clonal knockouts of known hypoxia response factors and utilizing whole-genome screens to identify novel genetic effectors. The second specific aim will investigate the direct metabolic consequences arising from hypoxic genetic reprogramming amidst RC dysfunction by combining genetic perturbations and liquid chromatography-mass spectrometry (LC-MS) metabolite profiling of cellular extracts and spent media over time. Together, these complementary approaches will 1) shed new insight into the endogenous mechanisms by which cells can protect against a faulty RC under exposure to low oxygen, 2) refine our understanding of the key cellular vulnerabilities resulting from RC dysfunction which may lead to pathology in a variety of contexts, and 3) propose new therapeutic targets that may be applicable generally to a wide spectrum of disorders exhibiting mitochondrial dysfunction.